Abstract
Uric acid and hypoxanthine are produced in the catabolism of purine. Abnormal urinary levels of these products are associated with many diseases and therefore it is necessary to have a simple and rapid method to detect them. Hence, we report a simple reverse phase high performance liquid chromatography (HPLC/UV) technique, developed and validated for simultaneous analysis of uric acid, hypoxanthine, and creatinine in human urine. Urine was diluted appropriately and eluted with C-18 column 100 mm × 4.6 mm with a C-18 precolumn 25 mm × 4.6 mm in series. Potassium phosphate buffer (20 mM, pH 7.25) at a flow rate of 0.40 mL/min was employed as the solvent and peaks were detected at 235 nm. Tyrosine was used as the internal standard. The experimental conditions offered a good separation of analytes without interference of endogenous substances. The calibration curves were linear for all test compounds with a regression coefficient, r2 > 0.99. Uric acid, creatinine, tyrosine, and hypoxanthine were eluted at 5.2, 6.1, 7.2, and 8.3 min, respectively. Intraday and interday variability were less than 4.6% for all the analytes investigated and the recovery ranged from 98 to 102%. The proposed HPLC procedure is a simple, rapid, and low cost method with high accuracy with minimum use of organic solvents. This method was successfully applied for the determination of creatinine, hypoxanthine, and uric acid in human urine.
Highlights
In purine catabolism, xanthine oxidase oxidizes hypoxanthine to xanthine and xanthine to uric acid
The abnormal levels of uric acid in serum and urine are associated with defects in enzymes in purine metabolism [1, 2] and are reported in cardiovascular diseases, gouty arthritis, renal disease, type 2 diabetes, obesity, and hypertension [2,3,4,5,6,7,8]
The stock standards were prepared at a concentration of 500 μg/mL for each analyte, uric acid, creatinine, and hypoxanthine in 100 mL volumetric flasks with deionized water or urine
Summary
Xanthine oxidase oxidizes hypoxanthine to xanthine and xanthine to uric acid. The abnormal levels of uric acid in serum and urine are associated with defects in enzymes in purine metabolism [1, 2] and are reported in cardiovascular diseases, gouty arthritis, renal disease, type 2 diabetes, obesity, and hypertension [2,3,4,5,6,7,8]. The levels of creatinine have been used to standardize the concentration of urinary constituents to improve the accuracy of the analysis [10,11,12]. HPLC methods for simultaneous quantification of creatinine, uric acid, and hypoxanthine have been reported. HPLC analysis of uric acid and hypoxanthine in human urine has been. An internal standard was used to improve the accuracy in the quantification of analytes. The method developed was applied to determine urinary creatinine, uric acid, and hypoxanthine levels in children with autism
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